Karien Labuschagne

Comparison of the efficiency of five suction light traps under field conditions in South Africa for the collection of Culicoides species.

Culicoides biting midges (Diptera: Ceratopogonidae) are involved in the transmission of a variety of pathogens, the economically most important being the orbiviruses that cause bluetongue and African horse sickness; both of which have been shown to be multi-vector diseases. The identification of all potential vectors will be crucial for the implementation of integrated control measures and disease risk analysis. The primary monitoring tools used for the collection of Culicoides midges are various models of suction light traps. In order to facilitate comparison of data between laboratories the efficiency of five traps (Onderstepoort, Rieb, mini-CDC, Pirbright, BG-sentinel), used at present and in the past in Europe, was compared in the field in South Africa. Comparisons were done either in three replicates of a 4x4 or two replicates of a 5x5 randomized Latin square design. The Onderstepoort trap collected significantly more Culicoides midges than the other traps. Relatively small but statistically significant differences were found in the species composition, parous rates, sex ratios as well as the ratio of Culicoides midges to other insects, as determined by the different traps. It will be important to determine the significance and underlying causes for these differences.

Life-history parameters of Culicoides (Avaritia) imicola Kieffer in the laboratory at different rearing temperatures.

This laboratory study investigates the sub-adult developmental cycle of field collected Culicoides (Avaritia) imicola Kieffer (Diptera; Ceratopogonidae). The period required from blood-feeding field-collected females to the production of progeny adults occupied 34-56 days at 20 degrees C, 15-21 days at 25 degrees C and 11-16 days at 28 degrees C, demonstrating clear temperature dependence. When reared at 28 degrees C, C. imicola demonstrated higher variability in fecundity (between 2.4 and 20.6 eggs/female) and lower hatching rates (50.0-62.2%), although larval survival rates to pupation were low at all temperatures (20-30%). Similarly, the mean emergence rate from pupae was the highest at lower temperatures. These results highlight the difficulty in establishing and maintaining a laboratory colony of this species from field-collected material and results are discussed in reference to future research directions that may aid this process.

Comparison of two trapping methods for Culicoides biting midges and determination of African horse sickness virus prevalence in midge populations at Onderstepoort, South Africa

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of a variety of pathogens including African horse sickness virus (AHSV), a member of the family Reoviridae, genus Orbivirus. AHSV causes African horse sickness (AHS), an endemic disease of equids with an extremely high mortality rate in horses in sub-Saharan Africa. Culicoides (Avaritia) imicola Kieffer is considered to be the principal vector of AHSV and is the dominant Culicoides species in South Africa. Due to the global distribution of Culicoides vectors, there is a potential risk of AHS spreading from endemic areas to areas traditionally free of the disease, which could have a severe economical impact on the affected equine industry. As part of any risk assessment it is essential to monitor known vectors as well as potential vector species. In the present study, sampling of Culicoides insects was compared using overnight collections in the conventional Onderstepoort light trap and mechanical aspiration of midges at sunset from bait horses. Culicoides imicola was confirmed as the predominant species using both trapping methods. Other species, mainly Culicoides (Avaritia) bolitinos Meiswinkel and Culicoides (Avaritia) gulbenkiani Caeiro, were highly underrepresented in the light trap collections, but made a significant contribution to the mechanical aspiration catches. The time for optimal collection differed between the trapping methods, leading to the conclusion that mechanical aspiration is a useful addition to conventional light trap collection and possibly the better choice when investigating insect vectors. An infection rate of 1.14% was calculated for the midge population based on real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays of collected Culicoides midges, which exceeds previous estimates. This is probably due to the increased sensitivity of the RT-qPCR assay used in this study as compared to the virus isolation assays used in previous studies. RT-qPCR-positive midges were present in midge pools obtained from both light trap and mechanical aspiration. Seven of the positive pools consisted of C. imicola only, four contained mixed species and one pool contained no C. imicola, suggesting the presence of AHSV in midges of other species.

First record of Culicoides oxystoma Kieffer and diversity of species within the Schultzei group of Culicoides Latreille (Diptera: Ceratopogonidae) biting midges in Senegal

The Schultzei group of Culicoides Latreille (Diptera: Ceratopogonidae) is distributed throughout Africa to northern Asia and Australasia and includes several potential vector species of livestock pathogens. The taxonomy of the species belonging to this species group is confounded by the wide geographical distribution and morphological variation exhibited by many species. In this work, morphological and molecular approaches were combined to assess the taxonomic validity of the species and morphological variants of the Schultzei group found in Senegal by comparing their genetic diversity with that of specimens from other geographical regions. The species list for Senegal was updated with four species: Culicoides kingi, C. oxystoma, C. enderleini and C. nevilli being recorded. This is the first record of C. oxystoma from Africa south of Sahara, and its genetic relationship with samples from Israel, Japan and Australia is presented. This work provides a basis for ecological studies of the seasonal and spatial dynamics of species of this species group that will contribute to better understanding of the epidemiology of the viruses they transmit.

Revision of the Culicoides (Avaritia) Imicola complex Khamala & Kettle (Diptera: Ceratopogonidae) from the Australasian region.

The monophyly of the Imicola complex, a natural species complex within subgenus C. subgen. Avaritia Fox of the biting midge genus Culicoides Latreille, is supported using morphological and molecular analyses. A diagnosis for the group along with comparative redescriptions of the male and female of the species represented in Australasia, C. brevitarsis Kieffer and C. nudipalpis Delfinado and a description of C. asiatica Bellis sp. nov., are presented together with keys for their specific determination and molecular support for their status.

The attraction range of the Onderstepoort 220 V light trap for Culicoides biting midges as determined under South African field conditions

Despite some limitations suction light traps are the primary tools used for the collection of Culicoides species (Diptera: Ceratopogonidae). The range of attraction of the Onderstepoort light trap is not known but an insight into the range of a trap will determine where the trap must be positioned relative to the hosts present, possible breeding sites and environmental structures in the trapping vicinity. It will therefore contribute to a more meaningful interpretation and comparison of results between trapping events. In the present study the number of Culicoides midges collected in a single trap was compared to those of traps made with an additional trap respectively 1m, 4m and 8.5m away from the first. Treatments between sites were rotated in three replicates of a 4×4 Latin square design. While interactions were found in traps 4m apart no statistically significant interactions were found when they were 8.5m apart. The range of attraction, indicated by the interaction between two traps, will be between 2m and 4m. In interpreting light trap results the limitations of this collection method needs to be taken into consideration.

Duration of repellency of N,N-diethyl-3-methylbenzamide, citronella oil and cypermethrin against Culicoides species when applied to polyester mesh.

The repellent efficacy of 15% N,N-diethyl-3-methylbenzamide (DEET), 0.6% citronella oil, and 0.3% alpha-cyano-cypermethrin against Culicoides species was compared in three 5x5 Latin squares (15 replicates) under South African field conditions. DEET, citronella oil or alpha-cyano-cypermethrin were applied to polyester meshes that were fitted to down-draught suction 220V UV light traps which were operated overnight. No significant repellent effect against Culicoides was found for the citronella oil or the alpha-cyano-cypermethrin. DEET had a significant repellent effect against Culicoides species and C. imicola for all catches made from after sunset to before sunrise.

Colonization of the Mediterranean Basin by the vector biting midge species #Culicoides imicola#: an old story

Understanding the demographic history and genetic make‐up of colonizing species is critical for inferring population sources and colonization routes. This is of main interest for designing accurate control measures in areas newly colonized by vector species of economically important pathogens. The biting midge Culicoides imicola is a major vector of orbiviruses to livestock. Historically, the distribution of this species was limited to the Afrotropical region. Entomological surveys first revealed the presence of C. imicola in the south of the Mediterranean basin by the 1970s. Following recurrent reports of massive bluetongue outbreaks since the 1990s, the presence of the species was confirmed in northern areas. In this study, we addressed the chronology and processes of C. imicola colonization in the Mediterranean basin. We characterized the genetic structure of its populations across Mediterranean and African regions using both mitochondrial and nuclear markers, and combined phylogeographical analyses with population genetics and approximate Bayesian computation. We found a west/east genetic differentiation between populations, occurring both within Africa and within the Mediterranean basin. We demonstrated that three of these groups had experienced demographic expansions in the Pleistocene, probably because of climate changes during this period. Finally, we showed that C. imicola could have colonized the Mediterranean basin in the Late Pleistocene or Early Holocene through a single event of introduction; however, we cannot exclude the hypothesis involving two routes of colonization. Thus, the recent bluetongue outbreaks are not linked to C. imicola colonization event, but rather to biological changes in the vector or the virus.

Environmental drivers of Culicoides phenology: how important is species-specific variation when determining disease policy?

Since 2006, arboviruses transmitted by Culicoides biting midges (Diptera: Ceratopogonidae) have caused significant disruption to ruminant production in northern Europe. The most serious incursions involved strains of bluetongue virus (BTV), which cause bluetongue (BT) disease. To control spread of BTV, movement of susceptible livestock is restricted with economic and animal welfare impacts. The timing of BTV transmission in temperate regions is partly determined by the seasonal presence of adult Culicoides females. Legislative measures therefore allow for the relaxation of ruminant movement restrictions during winter, when nightly light-suction trap catches of Culicoides fall below a threshold (the ‘seasonally vector free period’: SVFP). We analysed five years of time-series surveillance data from light-suction trapping in the UK to investigate whether significant inter-specific and yearly variation in adult phenology exists, and whether the SVFP is predictable from environmental factors. Because female vector Culicoides are not easily morphologically separated, inter-specific comparisons in phenology were drawn from male populations. We demonstrate significant inter-specific differences in Culicoides adult phenology with the season of Culicoides scoticus approximately eight weeks shorter than Culicoides obsoletus. Species-specific differences in the length of the SVFP were related to host density and local variation in landscape habitat. When the Avaritia Culicoides females were modelled as a group (as utilised in the SFVP), we were unable to detect links between environmental drivers and phenological metrics. We conclude that the current treatment of Avaritia Culicoides as a single group inhibits understanding of environmentally-driven spatial variation in species phenology and hinders the development of models for predicting the SVFP from environmental factors. Culicoides surveillance methods should be adapted to focus on concentrated assessments of species-specific abundance during the start and end of seasonal activity in temperate regions to facilitate refinement of ruminant movement restrictions thereby reducing the impact of Culicoides-borne arboviruses.

The effect of 1-octen-3-ol and 4-methylphenol on Culicoides midge numbers collected with suction light traps in South Africa

Despite some shortcomings, suction light traps are the primary monitoring tool for the collection of Culicoides species (Diptera: Ceratopogonidae). Factors that may increase the efficiency of these traps need to be investigated. In the present study the numbers of Culicoides midges collected with two Onderstepoort black light traps baited with a mixture of 1-octen-3-ol and 4-methylphenol, as a potential olfactory cue, were compared to those of two unbaited traps. Comparisons were done in two and three replicates of a 4 × 4 randomized Latin square design in the presence and absence of cattle. The addition of 1-octen-3-ol and 4-methylphenol, released at 9.1 and 15.5mg/h, respectively, did not influence species richness, numbers collected, sex ratios or age-grading results. Comparisons of Culicoides numbers and especially the abundance of Culicoides imicola Kieffer in collections done in the presence and absence of cattle confirm previous findings that show that host animals will be the primary attraction for Culicoides midges and that light traps mostly sample midges already in the near vicinity of the host.